Post-translation modifications (PTMs) of proteins are responsible for a host of critical functions, ranging from accelerating protein folding to mediating protein-protein interactions.(1) Protein “glycation” is a non-enzymatic process for PTM formation wherein protein side-chains react spontaneously with open chain tautomers of carbohydrates. Mounting evidence suggests that protein glycation adducts, also called “advanced glycation end-products” or “AGEs”, are critically involved in both healthy and disease processes, including inflammation, diabetes, cancer, and normal human aging.(2, 3) Notably, AGEs often possess highly complex chemical structures, impeding their detailed chemical and biological characterization.(4) 
Glucosepane (1) is an important member of the AGE family that is both biologically and chemically significant (See FIG. 1). The molecule is formed as a “crosslink” from reaction sequences between arginine and lysine side-chains and one equivalent of hexose carbohydrate (most commonly glucose). Glucosepane is present on long-lived plasma proteins in the human body, such as collagen and lens crystallin,(3, 5) and is also found in high levels in various dietary sources, especially alkali-treated baked goods.(6) Researchers have speculated that glucosepane is directly involved in the pathophysiology of various conditions (e.g., diabetes, diabetes-related complications, and aging) due to patterns of glucosepane formation on disease-associated proteins. For example, analysis of skin biopsies obtained through the Diabetes Control and Complications Trial (DCCT) has determined that increases in skin glucosepane levels represent a significant, independent risk factor for the onset of diabetic nephropathy, retinopathy, and neuropathy.(3, 7) Additional studies have demonstrated that non-enzymatic glucosepane crosslinks outnumber enzyme-catalyzed crosslinks in human collagen in people over 65 years of age.(8) By age 100, glucosepane levels reach 2 nmol/mg collagen, which is almost ten times normal levels, whereas levels in diabetic patients can achieve up to twenty times those in healthy controls.(9, 10) 
Several mechanisms have been proposed for glucosepane's involvement in disease complications. For example, researchers have hypothesized that glucosepane modification can decrease protein turnover rate and impair the renewal of damaged proteins. Glucosepane crosslinks may also be responsible for reported age- and diabetes-related decreases in collagen digestibility.(3),(11, 12),(13) Others have speculated that glucosepane-induced Arg modification can decrease the number of integrin binding sites in collagen, causing endothelial cell apoptosis, extracellular matrix deposition, and basement membrane thickening.(14) Glucosepane may also serve as ligand for pattern recognition receptors such as RAGE.(15) leading to chronic inflammation, or as a neoepitope that drives the breaking of self-tolerance against modified extracellular matrix proteins, serving as a trigger for the induction of autoimmune processes. Finally, due to high levels of glucosepane and other AGEs in the human diet, it has been suggested that these materials may function as uremic toxins, leading to complications in the setting of renal failure.(16)
Despite glucosepane's health implications, biological investigations have been hampered by a scarcity of chemically homogeneous material available for study. Its complex non-enzymatic biosynthesis involves serial tautomerizations of Amadori adduct 4 to provide glucosone 3 (a process termed “carbonyl mobility”, FIG. 1B). During this process, each stereocenter undergoes epimerization, and therefore the glucosepane core exists in nature as a mixture of all eight possible diastereomers.(3),(17) These stereoisomers can only be chromatographically resolved into four binary mixtures, each containing two spectroscopically indistinguishable diastereomers with the same relative configuration at the 6, 7, and 8a positions, but opposite absolute configurations with respect to the enantiomerically pure backbone amino acids.(17) Despite significant effort, purification of stereochemically homogeneous glucosepane from biological samples has proven impossible. It is therefore unknown which of the eight stereoisomers is the most prevalent in vivo. Furthermore, these binary diastereomeric mixtures can only be isolated in low yields (0.2-1.4%) following model reactions between lysine, arginine and glucose, and extensive chromatographic purification.(17, 18) Importantly, because of these difficulties in purification, antibody reagents to enable biological detection of glucosepane in unprocessed tissue preparations are unavailable. To our knowledge, therefore, all published investigations into glucosepane biology have relied upon time-consuming extraction protocols, involving exhaustive enzymatic hydrolysis followed by HPLC purification. The development of synthetic routes toward chemically-defined glucosepane constructs represents an essential next step toward understanding the roles that this compound plays in human health and disease, and also toward the identification of novel therapeutic and/or diagnostic agents.
Glucosepane presents a deceptively challenging synthetic target due to its high density of heteroatoms, the presence of a stereogenic polyol motif incorporated within a fused hetero-bicyclic topology, an epimerizable stereocenter at C-8a, and perhaps most notably, the presence of an arginine-derived iso-imidazole at its core. Indeed, at first glance, one would expect glucosepane to tautomerize spontaneously to the corresponding aromatic imidazole (FIG. 1A); however, reported structural assignments of the iso-imidazole in glucosepane are consistent with one- and two-dimensional NMR data reported by Lederer and colleagues.(17) Furthermore, because glucosepane forms naturally as a protein adduct (not as the free bis-amino acid crosslink), any useful synthesis needs to be compatible with glucosepane incorporation into peptides. Also, because glucosepane is formed naturally as a mixture of all eight possible diastereomers, synthetic efforts targeting both enantio- and diastereomerically pure material are essential for detailed biochemical study.